High affinity binding of human vitamin K-dependent protein S to a truncated recombinant beta-chain of C4b-binding protein expressed in Escherichia coli.

C4b-binding protein (C4BP) is a plasma glycoprotein that contains independent binding sites for complement component C4b and anticoagulant vitamin K-dependent protein S. C4BP is composed of seven alpha-chains (70 kDa) and one beta-chain (45 kDa) joined by disulfide bonds. In addition to non-repeat regions, the alpha- and beta-chains contain eight and three tandemly arranged modules, respectively, designated short consensus repeats (SCRs). C4b binds to the alpha-chains while it has been suggested, though not conclusively shown, that the beta-chain binds protein S. A truncated recombinant beta-chain composed of the three SCRs was expressed as a fusion protein with an epitope for a calcium-dependent monoclonal antibody (HPC4) in a procaryotic expression system. A signal peptide from the pelB gene of Erwinia carotovora directed expression to the medium from which the recombinant protein was purified using a HPC4 affinity column. On unreduced polyacrylamide gel electrophoresis in the presence of SDS, the recombinant protein demonstrated two closely spaced bands (M(r) = 19,000 and 20,000), whereas after reduction a broad band with an apparent molecular weight of 25,000 was obtained. On 125I-protein S ligand blotting, only one of the unreduced bands was found to bind protein S. The protein corresponding to this band bound protein S with almost as high affinity as intact C4BP, and it completely inhibited binding of protein S to intact C4BP. After reduction, the protein S binding ability was lost. The multiple carbohydrate side chains present in the native beta-chain of C4BP were apparently not required for protein S binding, as the recombinant beta-chain was not glycosylated. These results demonstrate that the protein S binding site of C4BP is contained within the three SCR modules of its beta-chain.